Surface immobilization of biomolecules is a very important step in the manufacturing of biosensors, microbeads, biochips, probe arrays, medical implants, and other devices. The key requirements for this process are the preservation of biochemical properties of immobilized substrates and robustness of the linkage. Copper (I)-catalyzed Huisgen 1,3-dipolar cycloaddition of azides to terminal acetylenes has emerged as one of the most convenient methods for the functionalization of various surfaces. The triazol linker fowled in the azide “click” reaction has excellent chemical stability due to the aromatic character of the formed heterocycle. Azide tags can be incorporated into biomolecules using a variety of different strategies, such as post-synthetic modification, in vitro enzymatic transfer, the use of covalent inhibitors, and metabolic labeling by feeding cells a biosynthetic precursor modified with azido functionality. While conventional copper (I)-catalyzed click chemistry has become commonplace in surface derivatization, as well as polymer and materials synthesis, the use of metal catalyst often limits the utility of the method. Copper ions are cytotoxic, can cause degradation of DNA molecules, and induce protein denaturation. In addition, the use of catalysts complicates kinetics of the immobilization process, requires polar solvents, and can alter surface properties.
Conventional azide click coupling methods employ terminal acetylenes, since internal alkynes react with azides only at elevated temperatures. Cyclooctynes, on the other hand, are known to form triazoles without a catalyst under ambient conditions, albeit at rather slow rate. The triple bond incorporated into an eight-membered ring is apparently already bent into a geometry resembling the transition state of the cycloaddition reaction, thus reducing its activation barrier. Recently-developed cyclooctyne derivatives are substantially more reactive towards azides and offer a convenient metal-free alternative to the copper-catalyzed click reaction (e.g., Jewett et al., J. Am. Chem. Soc. 2010, 132:3688-3690; Ning et al., Angew. Chem., Int. Ed. 2008, 47:2253-2255; and Debets et al., Chem. Commun. 2010, 46:97-99). Metal-free click chemistry has been successfully employed for the modification of luminescent quantum dots, proteins labeling and purification, as well as for the introduction of fluorescent tags into live cells (Poloukhtine et al., J. Am. Chem. Soc. 2009, 131:15769-15776) and organisms.
There remains a continuing need for new materials and methods for coupling azides to alkynes.